Vaccine and method for treatment of neurodegenerative diseases

ABSTRACT

Methods and compositions are provided for treatment of neurodegenerative diseases in which there is accumulation of misfolded and/or aggregated proteins, excluding prion diseases. In particular, the invention relates to treatment of the neurodegenerative diseases Huntington&#39;s disease (HD), Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD), by administration of an agent selected from the group consisting of (i) Copolymer 1, (ii) a Copolymer 1-related peptide, (iii) a Copolymer 1-related polypeptide, and (iv) T cells activated with (i), (ii) or (iii).

FIELD OF THE INVENTION

The present invention relates to compositions, e.g. vaccines, andmethods for the treatment of neurodegenerative diseases in which thereis accumulation of misfolded and/or aggregated proteins, excluding priondiseases. In particular, the invention relates to treatment of theneurodegenerative diseases Huntington's disease (HD), Alzheimer'sdisease (AD) or Parkinson's disease (PD), by administration of an agentselected from the group consisting of Copolymer 1, a Copolymer 1-relatedpeptide or polypeptide, and T cells activated therewith.

ABBREVIATIONS

Aβ₁₋₄₀, β-amyloid peptide 1-40; AD, Alzheimer's disease; APC,antigen-presenting cell; CNS, central nervous system; Cop-1, Copolymer1; DAT, dopamine transporter; HD, Huntington's disease; IRPB,interphotoreceptor retinoid-binding protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; OHSC, organotypic hippocampalslice culture; PD, Parkinson's disease; PI, propidium iodide; RGC,retinal ganglion cell; Treg, CD4⁺CD25⁺regulatory T cells; TUNEL,terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling;WRH, whole retinal homogenate.

BACKGROUND OF THE INVENTION

Pathological Disorders of the CNS Involving Accumulation of Misfoldedand/or Aggregated Proteins

For many decades, clinicians have been aware of the formation ofinsoluble protein aggregates in particular diseases. In Alzheimerdisease (Selkoe, 1997, 2002), the presence in the CNS ofβ-amyloid-containing plaques is associated with neurodegeneration anddementia. Similarly, other neurodegenerative diseases have recently beendiscovered to involve protein aggregation in the brain. For example,prion diseases such as kuru, Creutzfeldt-Jacob disease and bovinespongiform encephalopathy are associated with amyloid deposits of theprion protein (PrP). Polyglutamine repeat diseases such as Huntingtondisease are likewise associated with neuronal cytosolic and intranuclearinclusions (DiFiglia et al., 1997). These inclusions are composed offibrils that stain similarly to amyloid (Scherzinger et al., 1997).Finally, in Parkinson disease, inclusions known as Lewy bodies, found inthe cytoplasm of cells of the basal ganglia, include amyloid-likeaggregates of the protein α-synuclein (Conway et al., 2000; Serpell etal., 2000).

Huntington's disease (HD), identified in the late 1800s by the physicianGeorge Huntington, is an autosomal dominant neurodegenerative diseasewhose symptoms are caused by the loss of cells in the basal ganglia ofthe brain. This damage to cells affects cognitive ability (thinking,judgment, memory), movement, and emotional control. HD is characterizedby uncontrollable, dancelike movements and personality changes. HDpatients develop slurred speech, an unsteady walk and difficulty inswallowing. There is no effective treatment for HD. After a longillness, individuals with HD die from complications such as choking orinfection.

In 1993, the mutation that causes HD was identified as an unstableexpansion of CAG repeats in the IT15 gene encoding huntingtin, a proteinof unknown function (Menalled and Chesselet, 2002). The CAG repeatexpansion results in an increased stretch of glutamines in theN-terminal portion of the protein, which is widely expressed in brainand peripheral tissues (Gutekunst et al., 1995). The exact mechanismsunderlying neuronal death in Huntington's disease remain unknown.Proposed mechanisms have included activation of caspases or othertriggers of apoptosis, mitochondrial or metabolic toxicity, andinterference with gene transcription. Recent advances in theunderstanding of the pathophysiology of neurodegenerative diseases ingeneral and of Huntington's disease in particular, have suggested newtherapeutic strategies aimed at slowing progression or delay onset ofthe neurodegenerative disease.

Alzheimer's disease (AD) is an irreversible, progressive brain disorderthat occurs gradually and results in memory loss, behavioral andpersonality changes, and a decline in mental abilities. These losses arerelated to the death of brain cells and the breakdown of the connectionsbetween them. The course of this disease varies from person to person,as does the rate of decline. On average, AD patients live for 8 to 10years after they are diagnosed, though the disease can last up to 20years.

AD advances by stages, from early, mild forgetfulness to a severe lossof mental function. At first, AD destroys neurons in parts of the brainthat control memory, especially in the hippocampus and relatedstructures. As nerve cells in the hippocampus stop functioning properly,short-term memory fails. AD also attacks the cerebral cortex,particularly the areas responsible for language and reasoningEventually, many other areas of the brain are involved.

Parkinson's disease (PD) is an idiopathic, slowly progressive,degenerative CNS disorder characterized by slow and decreased movement,muscular rigidity, resting tremor, and postural instability. Despiteextensive investigations, the cause of PD remains unknown. The loss ofsubstantia nigra neurons, which project to the caudate nucleus andputamen, results in the depletion of the neurotransmitter dopamine inthese areas. Significant hints into PD pathogenesis have been yielded bythe use of 1-methyl-4-phenyl-1,2,4,6-tetrandropyridine (MPTP), aneurotoxin that replicates most of the neuropathological hallmarks of PDin humans, nonhuman primates, and other mammalian species, includingmice. Although the MPTP mouse model departs from human PD in a fewimportant ways, it offers a unique means to investigate, in vivo,molecular events underlying the demise of midbrain dopaminergic neurons(Dauer and Przedborski, 2003).

Acute and/or chronic neuronal loss in the adult CNS results in theirreversible loss of function due to the very poor ability of maturenerve cells to proliferate and compensate for the lost neurons. Thusattenuating or reducing neuronal loss is essential for preservation offunction. In most of the neurodegenerative diseases like Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) andHuntington's disease, the etiology is not clear, hence they areincurable. Nevertheless, there are some primary and secondary riskfactors, which are the target for therapeutic intervention aiming atinhibiting or attenuating progress of neuronal loss, collectively termedas neuroprotective therapy. Some of the risk factors aredisease-specific but others, like excitatory amino acids, free radicalsand nitric oxide, are common to all the neurodegenerative disorders.These factors are essential self-components in the healthy CNS, but withtheir accumulation in excess amounts in the degenerative tissue, theybecome cytotoxic leading to the spread of damage beyond the initialcause of neuron death.

Glutamate is one of the most common mediators of toxicity in acute andchronic degenerative disorders like status epilepticus, cerebralischemia, traumatic brain injury, ALS, Huntington's disease, lathyrismsand Alzheimer's disease. Glutamate is a primary excitatoryneurotransmitter in the human CNS. L-glutamate is present at a majorityof synapses and is capable of displaying dual activity: it plays apivotal role in normal functioning as an essential neurotransmitter, butbecomes toxic when its physiological levels are exceeded.

In order to minimize neuronal loss (neuroprotection) several approacheshave been adopted, at which the most common is targeting the riskfactors in an attempt to neutralize or inhibit their action.Unfortunately, these therapeutic strategies showed marginal efficacy inhuman subjects with concomitant severe side effects. The failure ofagents with discrete singular mechanisms of action argues for amulti-pronged approach.

Protective Autoimmunity

Loss of neurons in patients with devastating chronic neurodegenerativedisorders is attributed to numerous factors, most of them (for example,oxidative stress, ion imbalance, metabolic deficits, neurotransmitterimbalance, neurotoxicity) common to all such diseases (Doble, 1999).Even those factors that are apparently unique to a particular disordershare certain common features, including changes in the extracellulardeposition of self-compounds resulting in conformational and otherchanges, as well as in their aggregation, often culminating in plaqueformation (Hardy and Selkoe, 2002).

The local immune response to injuries in the CNS has often been blamedfor the progressive neurodegeneration that occurs after an insult(Hauben and Schwartz, 2003). Studies in the inventors' laboratory,however, have challenged the long-held notion that activated microgliaor blood-borne activated macrophages contribute to the ongoingpathology, and suggest instead that these immune cells are harnessed toaid recovery, but may be unable to display a significant positive effectbecause they fail to acquire the necessary phenotype (activity) orbecause their intervention is not strong enough or is inappropriatelytimed. This suggestion was supported by the demonstration that, in rats,macrophages activated by peripheral nerve (Rapalino et al., 1998) or byskin (Bomstein et al., 2003) can be helpful in promoting recovery fromspinal cord injury. The functional activity of such macrophages wasrecently found to resemble that of APC (Bomstein et al., 2003).

Subsequent studies by the inventors suggested that after a mechanical orbiochemical insult to the CNS the local immune response, which ismediated by T cells directed against self-antigens residing in the siteof the lesion (i.e., autoimmune T cells), determines the ability of theneural tissue to withstand the unfriendly extracellular conditionsresulting from the injury. It thus seems that the body protects itselfagainst toxic self-compounds in the CNS by harnessing a peripheraladaptive immune response in the form of T cells specific to antigensresiding in the site of damage (Hauben et al., 2000a; Moalem et al.,1999a; Yoles et al., 2001; Schori et al., 2001a; Schori et al., 2001b).The T cells that mediate protection are directed not against aparticular threatening self-compound but rather against dominantself-antigens that reside at the lesion site (Mizrahi et al., 2002;Schwartz et al., 2003; Bakalash et al., 2002).

Further studies by the inventors suggested that T-cell specificity isneeded in order to ensure that among the T cells that arrive at thesite, those encountering their specific or cross-reactive antigens(presented to them by local microglia acting as APC) will becomeactivated. The activated T cells can then provide the necessarycytokines or growth factors that control the activity of the localmicroglia and the friendliness of the extracellular milieu (Schwartz etal., 2003; Moalem et al., 2000; Kipnis et al., 2000).

The concept of T cell-dependent “protective autoimmunity” has beenformulated by the inventor Prof. Michal Schwartz and her group (Kipniset al., 2002a; Schwartz and Kipnis, 2002a). According to this concept,an acute or chronic insult to the CNS triggers an autoimmune responsedirected against proteins residing in the lesion site. T cells homing tothe lesion site are activated by cells presenting the relevant antigen.Once activated, they augment and control local immune cells, allowingefficient removal of toxic compounds and tissue debris, thus protectingthe damaged nerves from further degeneration. The potential of theimmune system to counteract the hostile conditions is enhanced byboosting the normal immune response. Based on this hypothesis, boostingthe immune system with a suitable antigen should provideneuroprotection. Among suitable antigens identified by the presentinventors is Copolymer 1.

Copolymer 1

Copolymer 1, also called Cop 1, is a random non-pathogenic syntheticcopolymer, a heterogeneous mix of polypeptides containing the four aminoacids L-glutamic acid (E), L-alanine (A), L-tyrosine (Y) and L-lysine(K) in an approximate ratio of 1.5:4.8:1:3.6, but with no uniformsequence. Although its mode of action remains controversial, Cop 1clearly helps retard the progression of human multiple sclerosis (MS)and of the related autoimmune condition studied in mice, experimentalautoimmune encephalomyelitis (EAE). One form of Cop 1, known asglatiramer acetate, has been approved in several countries for thetreatment of multiple sclerosis under the trademark Copaxone® (TevaPharmaceutical Industries Ltd., Petach Tikva, Israel).

Vaccination with Cop 1 or with Cop 1-activated T cells have been shownby the present inventors to boost the protective autoimmunity, aftertraumatic CNS insult, thereby reducing further injury-induced damage,and can further protect CNS cells from glutamate toxicity. Reference ismade to Applicant's previous U.S. patent applications Ser. Nos.09/765,301 and 09/765,644 and corresponding published InternationalApplication Nos. WO 01/52878 and WO 01/93893, which disclose that Cop 1,Cop 1-related peptides and polypeptides and T cells activated therewithprevent or inhibit neuronal degeneration and promote nerve regenerationin the CNS or peripheral nervous system (PNS), and protect CNS cellsfrom glutamate toxicity.

Prof. Schwartz and colleagues have shown that Cop 1 acts as alow-affinity antigen that activates a wide range of self-reacting Tcells, resulting in neuroprotective autoimmunity that is effectiveagainst both CNS white matter and grey matter degeneration (Kipnis etal., 2002a; Schwartz and Kipnis, 2002a). The neuroprotective effect ofCop 1 vaccination was demonstrated by the inventors in animal models ofacute and chronic neurological disorders such as optic nerve injury(Kipnis et al., 2000), head trauma (Kipnis et al., 2003), glaucoma(Schori et al., 2001b), amyotrophic lateral sclerosis (Angelov et al.,2003) and in the applicant's patent applications WO 01/52878, WO01/93893 and WO 03/047500.

The use of Copolymer 1 for treatment of prion-related diseases isdisclosed in WO 01/97785. Gendelman and co-workers disclose that passiveimmunization with splenocytes of mice immunized with Cop 1 confersdopaminergic neuroprotection in MPTP-treated mice (Benner et al., 2004).

All patents and patent applications cited herein are hereby incorporatedby reference in their entirety as if fully disclosed herein.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a method for treating aneurodegenerative disorder or disease in which there is accumulation ofmisfolded and/or aggregated proteins, excluding prion-related diseases,said method comprising administering to an individual in need an agentselected from the group consisting of (i) Copolymer 1, (ii) a Copolymer1-related peptide, (iii) a Copolymer 1-related polypeptide, and (iv) Tcells activated with (i), (ii) or (iii).

In one embodiment, the invention relates to a method for reducingdisease progression, and/or protection from neurodegeneration and/orprotection from glutamate toxicity in a patient suffering from aneurodegenerative disorder or disease in which there is accumulation ofmisfolded and/or aggregated proteins, excluding prion-related diseases,which comprises administering to said patient a therapeutically activeamount of an agent selected from the group consisting of (i) Copolymer1, (ii) a Copolymer 1-related peptide, (iii) a Copolymer 1-relatedpolypeptide, and (iv) T cells activated with (i), (ii) or (iii).

In another embodiment, the invention relates to a method for reducingdisease progression, and/or protection from neurodegeneration and/orprotection from glutamate toxicity in a patient suffering from aneurodegenerative disorder or disease in which there is accumulation ofmisfolded and/or aggregated proteins, excluding prion-related diseases,which comprises immunizing said patient with an agent selected from thegroup consisting of (i) Copolymer 1, (ii) a Copolymer 1-related peptide,(iii) a Copolymer 1-related polypeptide, and (iv) T cells activated with(i), (ii) or (iii).

In another aspect, the present invention provides a pharmaceuticalcomposition for treatment of a neurodegenerative disorder or disease inwhich there is accumulation of misfolded and/or aggregated proteins,excluding prion-related diseases, comprising a pharmaceuticallyacceptable carrier and an active agent selected from the groupconsisting of (i) Copolymer 1, (ii) a Copolymer 1-related peptide, (iii)a Copolymer 1-related polypeptide, and (iv) T cells activated with (i),(ii) or (iii). In one embodiment, said pharmaceutical composition is avaccine.

In a further aspect, the present invention relates to the use of anactive agent selected from the group consisting of (i) Copolymer 1, (ii)a Copolymer 1-related peptide, (iii) a Copolymer 1-related polypeptide,and (iv) T cells activated with (i), (ii) or (iii), for the manufactureof a pharmaceutical composition for treatment of a neurodegenerativedisorder or disease in which there is accumulation of misfolded and/oraggregated proteins, excluding prion-related diseases. In oneembodiment, said pharmaceutical composition is a vaccine.

In one embodiment, said neurodegenerative disease or disorder isHuntington's disease. In another embodiment, said neurodegenerativedisease or disorder is Alzheimer's disease. In a further embodiment,said neurodegenerative disease or disorder is Parkinson's disease.

In still another aspect, the invention provides an article ofmanufacture comprising packaging material and a pharmaceuticalcomposition contained within the packaging material, said pharmaceuticalcomposition comprising an agent selected from the group consisting ofCopolymer 1, a Copolymer 1-related peptide, and a Copolymer 1-relatedpolypeptide; and said packaging material includes a label that indicatesthat said agent is therapeutically effective for treating aneurodegenerative disease or disorder selected from Huntington'sdisease, Alzheimer's disease or Parkinson's disease.

In the most preferred embodiment of the invention, the active agent isCopolymer 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the neuroprotective effect on retinal ganglion cells (RGCs)of mice by immunization with different doses of Cop 1 (25, 75 g or 225μg/mouse) injected 7 days before exposure of RGCs to glutamate toxicity.The results are presented as mean±SEM of percentage of RGCs that wereprotected due to Cop 1 vaccination out of the total RGC death in thenon-treated group. * represents statistically significant difference(t-test, p<0.05) versus the non-treated group.

FIG. 2 shows the latency of neuroprotective effect on RGCs of mice byvaccination with 75 μg Cop 1 injected 7, 14 and 28 days before exposureof RGCs to glutamate toxicity.

FIG. 3 shows that daily injections of Cop 1 repeated for three days atdoses of 25 μg and 75 μg, cause loss of the neuroprotective effect onRGCs (at 25 μg, protection of 23% and 1.5% after 2 and 3 days,respectively; at 75 μg, protection of 47% and 13.5% after 2 and 3 days,respectively).

FIG. 4 shows the efficacy of two repeated injections of Cop 1 (75μg/mouse), injected at different time intervals (1, 2, 3, 4, 6, 8,weeks). The neuroprotective effect of the treatment on RGCs isrepresented as % of a single injection of Cop 1 (75 μg/mouse), injected7 days before induction of glutamate toxicity. This single injection wasdetermined as positive control and performed in each experiment. *represents statistically significant difference (t-test, p<0.05) versusthe non-treated group.

FIG. 5 shows the efficacy of three repeated injections of Cop 1 (75μg/mouse) injected at different time intervals (daily, 1, 2, 4, weeks).The neuroprotective effect of the treatment on RGCs is represented as %of a single injection of Cop 1 (75 μg/mouse), injected 7 days beforeinduction of glutamate toxicity. This single injection was determined aspositive control and performed in each experiment.

FIG. 6 shows proliferation of splenocytes from mice followingimmunization with different doses of Cop 1 (25 μg, 75 μg, 225 μg). Theresults after 7, 14, 21 and 28 days are expressed as stimulation index(SI), where SI is the mean cpm of cells incubated in vitro with Cop 1divided by the mean cpm of cells incubated in vitro without Cop 1.

FIG. 7 shows INF-γ secretion from stimulated splenocytes 7, 14, 21 or 28days after immunization with 25 μg or 75 μg Cop 1.

FIG. 8 is a graph showing the rotarod performance of HD R6/2 transgenicmice after vaccination with 75 μg or 150 μg Cop 1.

FIG. 9 shows the rotarod performance of HD R6/2 transgenic micefollowing vaccination with 150 μg Cop 1, at different speeds of rotation(2, 5, 15 and 25 rpm).

FIGS. 10A-10D show that immunization of mice with retinal proteinsprotects retinal ganglion cells against glutamate toxicity. (A)TUNEL-positive cells in the RGC layers of C57B1/6J mice 48 h afterintravitreal injection of a toxic dose of glutamate. Sections (20 μmthick) were subjected to TUNEL staining, counterstained with propidiumiodide, and viewed by confocal microscopy to detect TUNEL-positivecells. A confocal image of a representative retina is shown. The arrowindicates TUNEL-positive cells in the RGC layer. Scale bar=200 μm. (B)C57B1/6J mice were immunized in the flank with 600 μg of whole retinalhomogenate (WRH) emulsified in CFA supplemented with 5 mg/ml ofMycobacterium tuberculosis. Six days later the mice were injectedintravitreally with glutamate (400 nmol). One week later surviving RGCswere counted. Significantly more RGCs survived in mice immunized withWRH/CFA than in mice immunized with PBS/CFA. The figure shows theresults, expressed as neuronal loss relative to the RGC population ofnormal retinas, of one representative experiment out of two independentexperiments (n=6-8 mice per experiment in each group; P<0.0001,two-tailed Student's t-test). (C, D) In another set of experiments micewere immunized in the flank with interphotoreceptor-binding protein(IRBP; 50 μg) or S-antigen (50 μg) emulsified in CFA supplemented with 5mg/ml of Mycobacterium tuberculosis. Control mice were immunized withPBS in CFA. Significantly more neurons were lost in the PBS/CFAimmunized group than in the IRBP/CFA immunized group (P<0.0001;two-tailed Student's t-test; n=6-8 mice in each group) or in theS-antigen-immunized group (P<0.0001; n=6-8 mice per group).

FIGS. 11A-11F show that susceptibility of retinal ganglion cells toAβ₁₋₄₀ toxicity is T cell-dependent. (A) C57BL/6/J mice were injectedintravitreally with 5 or 50 μM Aβ₁₋₄₀ or were not injected (control),and 1 or 2 weeks later the retinas were excised and the surviving RGCscounted. Relative to controls, significantly more neurons were lost inmice that had received the higher dose of Aβ₁₋₄₀ (P<0.001 and P<0.0001at 1 and 2 weeks, respectively, two-tailed Student's t-test). Valuesshown are from one of three independent experiments with similar results(n=6-8 mice per group). Injection of the vehicle resulted in a smallloss of RGCs relative to controls. (B) and (C), representativemicrographs of retinas injected or not injected with Aβ₁₋₄₀. (D)BALB/c/OLA mice [wild type or nu/nu (devoid of mature T cells)] wereinjected intravitreally with Aβ₁₋₄₀ (50 μM). Significantly more RGCswere lost in the nu/nu mice relative to the wild-type (P<0.001,two-tailed Student's t-test) Values shown are from one of threeindependent experiments with similar results (n=5-7 mice per group). (E)and (F), representative micrographs of retinas from wild-type BALB/c/OLAand nu/nu BALB/c/OLA mice injected with Aβ₁₋₄₀.

FIGS. 12A-12B show that immunization with an antigen residing in thesite of toxicity rather than with the toxic agent itself protectsagainst aggregated Aβ₁₋₄₀ toxicity in C57B1/6J mice. C57B1/6J mice wereimmunized in the flank with interphotoreceptor-binding protein (IRBP; 50μg) in CFA, the β-amyloid peptide (1-40, non-aggregated) (50 μg) in CFA,or PBS in CFA. In all cases, CFA was supplemented with 5 mg/ml ofMycobacterium tuberculosis. Ten days later the mice were injectedintravitreally with a toxic dose of aggregated Aβ₁₋₄₀ (50 μM), and after10 days their retinas were excised and the surviving RGCs counted. (A)Significantly fewer RGCs were lost in C57BL6/J mice immunized withIRBP/CFA than in matched controls treated with PBS/CFA (P<0.0008,two-tailed Student's t-test). (B) The mean number of surviving RGCs inmice immunized with native β-amyloid peptide in CFA did not differsignificantly from that in mice injected with PBS/CFA.

FIGS. 13A-13B show that passive transfer of activated splenocytes frommice immunized with dominant retinal antigens into naïve mice results inprotection. (A) Wild-type C57B1/6J mice were immunized in the hind footpads with a combination of interphotoreceptor-binding protein (IRBP) andS-antigen (50 μg each) or 50 μg OVA emulsified in CFA supplemented with5 mg/ml of Mycobacterium tuberculosis. Ten days later draining lymphnodes were excised and pooled, cell suspensions were prepared, and thecells were counted. Cells were activated ex-vivo by stimulation withtheir specific antigens for 48 h, and the activated T cells were theninjected i.p. into naïve C57B1/6J mice. T cells specific toIRBP+S-antigen were injected at a dose of 1.2×10⁷ T cells in PBS. Within1 h of passive T cell transfer the mice received an intravitrealinjection of glutamate (400 nmol), and surviving retinal ganglion cells(RGCs) were counted 1 week later. Significantly fewer RGCs survived inmice that received OVA-specific T cells than in mice that received Tcells specific to IRBP+S-antigen (P<0.001; two-tailed Student's t-test).There was no difference between mice that received OVA-specific T cellsand naïve mice in the numbers of RGCs that survived the glutamateinjection (n=4-6 mice per group). (B) Mice were injected intravenouslywith 8×10⁶ activated T cells directed either to IRBP or to β-amyloidpeptide (1-40, non-aggregated). One hour after this passive T-celltransfer, the mice were injected with a toxic dose of aggregated Aβ₁₋₄₀.Two weeks later their retinas were excised and surviving RGCs counted.Neuronal loss in these mice was significantly decreased by transfer of Tcells reactive to the IRBP (P<0.005, two-tailed Student's t-test), butwas not significantly affected by transfer of T cells reactive tonon-aggregated β-amyloid.

FIG. 14 shows that active immunization with Cop-1 protects againstAβ₁₋₄₀ toxicity. C57B1/6J mice were immunized with Cop-1, 6 days beforebeing injected intravitreally with aggregated Aβ₁₋₄₀. Two weeks latertheir retinas were excised and the surviving cells counted.Significantly fewer RGCs were lost in mice immunized with Cop-1 than inmatched controls treated with PBS (P<0.001, two-tailed Student'st-test).

FIGS. 15A-15C show that more neurons survive aggregated Aβ₁₋₄₀intoxication in mice devoid of naturally occurring regulatory CD4+CD25+T cells than in naïve mice. (A) C57B1/6J mice devoid of Treg as a resultof thymectomy 3 days after birth (TXD3 mice) were injectedintravitreally with a toxic dose of Aβ₁₋₄₀ at the age of 12 weeks.Significantly fewer RGCs were lost in the TXD3 mice than in age-matchednormal controls (P<0.001; two-tailed Student's t-test; n=6-8 mice pergroup). (B) BALB/c/OLA nu/nu mice were replenished with 4.5×10⁷splenocytes from spleens devoid of Treg or from whole spleens ofBALB/c/OLA mice. After injection of aggregated Aβ₁₋₄₀, significantlyfewer RGCs were lost in nu/nu mice replenished with splenocytes devoidof Treg than in matched wild-type controls (P<0.05; two-tailed Student'st-test). In both groups, significantly fewer RGCs were lost than inuntreated nu/nu mice injected with aggregated Aβ₁₋₄₀ (P<0.001;two-tailed Student's t-test). In each experiment, the number of RGCscounted in eyes not exposed to aggregated Aβ₁₋₄₀ toxicity was taken asthe normal baseline value. The results of one experiment out of two arepresented. (C) Semi-quantitative RT-PCR analysis of Foxp3 expression.mRNA was extracted from freshly isolated Teff and Treg. The housekeepinggene β-actin was used for quantitative analysis. The results shown arefrom one representative experiment out of five.

FIGS. 16A-16B show death of neural cells in rat organotypic hippocampalslice cultures 24 h after treatment with microglia incubated withaggregated Aβ₁₋₄₀ with and without activated T cells. OHSCs wereobtained from BALB/c/OLA mice. Immediately after sectioning, the sliceswere co-cultured for 24 h with microglia that had been pre-incubated (12h) with aggregated Aβ₁₋₄₀ alone or with a combination of aggregatedAβ₁₋₄₀ and activated Teff (A). Control slices were treated with naïvemicroglia or were left untreated. Twenty-four hours after co-culturingof microglia and brain slices, the slices were stained with propidiumiodide (PI) (a fluorescent dye that stains only dead cells) and analyzedby fluorescence microscopy. (A) quantification of PI intensity,calculated as a percentage of the intensity measured in untreatedcontrol OHSCs (*P<0.05; ** P<0.01, *** P<0.001; two-tailed Student'st-test; n=6-8 slices per group). (B), selected photomicrographs ofuntreated control slices (1), slices incubated with untreated microglia(2), slices treated with microglia that were pre-incubated withaggregated Aβ₁₋₄₀ (3), and slices treated with microglia that had beenexposed to aggregated Aβ₁₋₄₀ in conjunction with activated T cells (4).

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention comprise administering to anindividual in need an agent selected from the group consisting of (i)Copolymer 1, (ii) a Copolymer 1-related peptide, (iii) a Copolymer1-related polypeptide, and (iv) T cells activated with (i), (ii) or(iii), for the treatment of a neurodegenerative disorder or disease inwhich there is accumulation of misfolded and/or aggregated proteins,excluding prion-related diseases. In one preferred embodiment, theneurodegenerative disease or disorder is Huntington's disease. Inanother preferred embodiment, the neurodegenerative disease or disorderis Alzheimer's disease. In a further preferred embodiment, theneurodegenerative disease or disorder is Parkinson's disease.

The treatment with Copolymer 1, Cop 1-related peptides or polypeptides,of T cells activated therewith, aims to reduce disease progression, toafford protection from neurodegeneration, and/or to afford protectionfrom glutamate toxicity in patients suffering from Huntington's disease,Alzheimer's disease or Parkinson's disease. In one embodiment, thetreatment is performed by immunization. In another embodiment,therapeutically effective amounts of the selected agent are administeredto the patient. The doses and regimen of the two types of treatment maybe different.

Further provided by the present invention is a method for treating orpreventing neurodegeneration and cognitive decline and dysfunctionassociated with Huntington's disease, Alzheimer's disease orParkinson'sdisease, said method comprising administering to an individual in needan agent selected from the group consisting of (i) Copolymer 1, (ii) aCopolymer 1-related peptide, (iii) a Copolymer 1-related polypeptide,and (iv) T cells activated with (i), (ii) or (iii).

As used herein in the application, the terms “Cop 1” and “Copolymer 1”are used interchangeably.

For the purpose of the present invention, “Cop 1 or a Cop 1-relatedpeptide or polypeptide” is intended to include any peptide orpolypeptide, including a random copolymer, that cross-reactsfunctionally with myelin basic protein (MBP) and is able to compete withMBP on the MHC class II in the antigen presentation.

The composition or vaccine of the invention may comprise as active agenta Cop 1 or a Cop 1-related peptide or polypeptide represented by arandom copolymer consisting of a suitable ratio of a positively chargedamino acid such as lysine or arginine, in combination with a negativelycharged amino acid (preferably in a lesser quantity) such as glutamicacid or aspartic acid, optionally in combination with a non-chargedneutral amino acid such as alanine or glycine, serving as a filler, andoptionally with an amino acid adapted to confer on the copolymerimmunogenic properties, such as an aromatic amino acid like tyrosine ortryptophan. Such compositions may include any of those copolymersdisclosed in WO 00/05250, the entire contents of which are herewithincorporated herein by reference.

More specifically, the composition for use in the present inventioncomprises at least one copolymer selected from the group consisting ofrandom copolymers comprising one amino acid selected from each of atleast three of the following groups: (a) lysine and arginine; (b)glutamic acid and aspartic acid; (c) alanine and glycine; and (d)tyrosine and tryptophan.

The copolymers for use in the present invention can be composed of L- orD-amino acids or mixtures thereof. As is known by those of skill in theart, L-amino acids occur in most natural proteins. However, D-aminoacids are commercially available and can be substituted for some or allof the amino acids used to make the copolymers used in the presentinvention. The present invention contemplates the use of copolymerscontaining both D- and L-amino acids, as well as copolymers consistingessentially of either L- or D-amino acids.

In one embodiment of the invention, the copolymer contains fourdifferent amino acids, each from a different one of the groups (a) to(d).

In a more preferred embodiment, the pharmaceutical composition orvaccine of the invention comprises Copolymer 1, a mixture of randompolypeptides consisting essentially of the amino acids L-glutamic acid(E), L-alanine (A), L-tyrosine (Y) and L-lysine (K) in an approximateratio of 1.5:4.8:1:3.6, having a net overall positive electrical chargeand of a molecular weight from about 2 KDa to about 40 KDa. In onepreferred embodiment, the Cop 1 has average molecular weight of about 2KDa to about 20 KDa, more preferably of about 4.7 KDa to about 13 K Da,still more preferably of about 4 KDa to about 8.6 KDa, of about 5 KDa to9 KDa, or of about 6.25 KDa to 8.4 KDa. In another preferred embodiment,the Cop 1 has average molecular weight of about 13 KDa to about 20 KDa,more preferably of about 13 KDa to about 16 KDa or of about 15 KDa toabout 16 KDa. Other average molecular weights for Cop 1, lower than 40KDa, are also encompassed by the present invention. Copolymer 1 of saidmolecular weight ranges can be prepared by methods known in the art, forexample by the processes described in U.S. Pat. No. 5,800,808, theentire contents of which are hereby incorporated by reference in theentirety. The Copolymer 1 may be a polypeptide comprising from about 15to about 100, preferably from about 40 to about 80, amino acids inlength. In one preferred embodiment, the Cop 1 is in the form of itsacetate salt known under the generic name glatiramer acetate, that hasbeen approved in several countries for the treatment of multiplesclerosis (MS) under the trade name, Copaxone® (a trademark of TevaPharmaceuticals Ltd., Petach Tikva, Israel). The activity of Copolymer 1for the vaccine disclosed herein is expected to remain if one or more ofthe following substitutions is made: aspartic acid for glutamic acid,glycine for alanine, arginine for lysine, and tryptophan for tyrosine.

In another embodiment of the invention, the Cop 1-related peptide orpolypeptide is a copolymer of three different amino acids each from adifferent one of three groups of the groups (a) to (d). These copolymersare herein referred to as terpolymers.

In one embodiment, the Cop 1-related peptide or polypeptide is aterpolymer containing tyrosine, alanine, and lysine, hereinafterdesignated YAK, in which the average molar fraction of the amino acidscan vary: tyrosine can be present in a mole fraction of about0.05-0.250; alanine in a mole fraction of about 0.3-0.6; and lysine in amole fraction of about 0.1-0.5. More preferably, the molar ratios oftyrosine, alanine and lysine are about 0.10:0.54:0.35, respectively. Itis possible to substitute arginine for lysine, glycine for alanine,and/or tryptophan for tyrosine.

In another embodiment, the Cop 1-related peptide or polypeptide is aterpolymer containing tyrosine, glutamic acid, and lysine, hereinafterdesignated YEK, in which the average molar fraction of the amino acidscan vary: glutamic acid can be present in a mole fraction of about0.005-0.300, tyrosine can be present in a mole fraction of about0.005-0.250, and lysine can be present in a mole fraction of about0.3-0.7. More preferably, the molar ratios of glutamic acid, tyrosine,and lysine are about 0.26:0.16:0.58, respectively. It is possible tosubstitute aspartic acid for glutamic acid, arginine for lysine, and/ortryptophan for tyrosine.

In another preferred embodiment, the Cop 1-related peptide orpolypeptide is a terpolymer containing lysine, glutamic acid, andalanine, hereinafter designated KEA, in which the average molar fractionof the amino acids can vary: glutamic acid can be present in a molefraction of about 0.005-0.300, alanine in a mole fraction of about0.005-0.600, and lysine can be present in a mole fraction of about0.2-0.7. More preferably, the molar ratios of glutamic acid, alanine andlysine are about 0.15:0.48:0.36, respectively. It is possible tosubstitute aspartic acid for glutamic acid, glycine for alanine, and/orarginine for lysine.

In a preferred embodiment, the Cop 1-related peptide or polypeptide is aterpolymer containing tyrosine, glutamic acid, and alanine, hereinafterdesignated

YEA, in which the average molar fraction of the amino acids can vary:tyrosine can be present in a mole fraction of about 0.005-0.250,glutamic acid in a mole fraction of about 0.005-0.300, and alanine in amole fraction of about 0.005-0.800. More preferably, the molar ratios ofglutamic acid, alanine, and tyrosine are about 0.21: 0.65:0.14,respectively. It is possible to substitute tryptophan for tyrosine,aspartic acid for glutamic acid, and/or glycine for alanine.

The average molecular weight of the terpolymers YAK, YEK, KEA and YEAcan vary between about 2 KDa to 40 KDa, preferably between about 3 KDato 35 KDa, more preferably between about 5 KDa to 25 KDa.

Copolymer 1 and related peptides and polypeptides may be prepared bymethods known in the art, for example, under condensation conditionsusing the desired molar ratio of amino acids in solution, or by solidphase synthetic procedures. Condensation conditions include the propertemperature, pH, and solvent conditions for condensing the carboxylgroup of one amino acid with the amino group of another amino acid toform a peptide bond. Condensing agents, for exampledicyclohexylcarbodiimide, can be used to facilitate the formation of thepeptide bond. Blocking groups can be used to protect functional groups,such as the side chain moieties and some of the amino or carboxyl groupsagainst undesired side reactions.

For example, the copolymers can be prepared by the process disclosed inU.S. Pat. No. 3,849,550, wherein the N-carboxyanhydrides of tyrosine,alanine, γ-benzyl glutamate and N ε-trifluoroacetyl-lysine arepolymerized at ambient temperatures (20° C.-26° C.) in anhydrous dioxanewith diethylamine as an initiator. The γ-carboxyl group of the glutamicacid can be deblocked by hydrogen bromide in glacial acetic acid. Thetrifluoroacetyl groups are removed from lysine by 1 M piperidine. One ofskill in the art readily understands that the process can be adjusted tomake peptides and polypeptides containing the desired amino acids, thatis, three of the four amino acids in Copolymer 1, by selectivelyeliminating the reactions that relate to any one of glutamic acid,alanine, tyrosine, or lysine.

The molecular weight of the copolymers can be adjusted duringpolypeptide synthesis or after the copolymers have been made. To adjustthe molecular weight during polypeptide synthesis, the syntheticconditions or the amounts of amino acids are adjusted so that synthesisstops when the polypeptide reaches the approximate length which isdesired. After synthesis, polypeptides with the desired molecular weightcan be obtained by any available size selection procedure, such aschromatography of the polypeptides on a molecular weight sizing columnor gel, and collection of the molecular weight ranges desired. Thecopolymers can also be partially hydrolyzed to remove high molecularweight species, for example, by acid or enzymatic hydrolysis, and thenpurified to remove the acid or enzymes.

In one embodiment, the copolymers with a desired molecular weight may beprepared by a process, which includes reacting a protected polypeptidewith hydrobromic acid to form a trifluoroacetyl-polypeptide having thedesired molecular weight profile. The reaction is performed for a timeand at a temperature which is predetermined by one or more testreactions. During the test reaction, the time and temperature are variedand the molecular weight range of a given batch of test polypeptides isdetermined. The test conditions which provide the optimal molecularweight range for that batch of polypeptides are used for the batch.Thus, a trifluoroacetyl-polypeptide having the desired molecular weightprofile can be produced by a process, which includes reacting theprotected polypeptide with hydrobromic acid for a time and at atemperature predetermined by test reaction. Thetrifluoroacetyl-polypeptide with the desired molecular weight profile isthen further treated with an aqueous piperidine solution to form a lowtoxicity polypeptide having the desired molecular weight.

In a preferred embodiment, a test sample of protected polypeptide from agiven batch is reacted with hydrobromic acid for about 10-50 hours at atemperature of about 20-28° C. The best conditions for that batch aredetermined by running several test reactions. For example, in oneembodiment, the protected polypeptide is reacted with hydrobromic acidfor about 17 hours at a temperature of about 26° C.

As binding motifs of Cop 1 to MS-associated HLA-DR molecules are known(Fridkis-Hareli et al, 1999), polypeptides derived from Cop 1 having adefined sequence can readily be prepared and tested for binding to thepeptide binding groove of the HLA-DR molecules as described in theFridkis-Hareli et al (1999) publication. Examples of such peptides arethose disclosed in WO 00/05249 and WO 00/05250, the entire contents ofwhich are hereby incorporated herein by reference, and include thepeptides of SEQ ID NOs. 1-32 hereinbelow.

SEQ ID NO. Peptide Sequence 1 AAAYAAAAAAKAAAA 2 AEKYAAAAAAKAAAA 3AKEYAAAAAAKAAAA 4 AKKYAAAAAAKAAAA 5 AEAYAAAAAAKAAAA 6 KEAYAAAAAAKAAAA 7AEEYAAAAAAKAAAA 8 AAEYAAAAAAKAAAA 9 EKAYAAAAAAKAAAA 10 AAKYEAAAAAKAAAA11 AAKYAEAAAAKAAAA 12 EAAYAAAAAAKAAAA 13 EKKYAAAAAAKAAAA 14EAKYAAAAAAKAAAA 15 AEKYAAAAAAAAAAA 16 AKEYAAAAAAAAAAA 17 AKKYEAAAAAAAAAA18 AKKYAEAAAAAAAAA 19 AEAYKAAAAAAAAAA 20 KEAYAAAAAAAAAAA 21AEEYKAAAAAAAAAA 22 AAEYKAAAAAAAAAA 23 EKAYAAAAAAAAAAA 24 AAKYEAAAAAAAAAA25 AAKYAEAAAAAAAAA 26 EKKYAAAAAAAAAAA 27 EAKYAAAAAAAAAAA 28AEYAKAAAAAAAAAA 29 AEKAYAAAAAAAAAA 30 EKYAAAAAAAAAAAA 31 AYKAEAAAAAAAAAA32 AKYAEAAAAAAAAAA

Such peptides and other similar peptides derived from Cop 1 would beexpected to have similar activity as Cop 1. Such peptides, and othersimilar peptides, are also considered to be within the definition of Cop1-related peptides or polypeptides and their use is considered to bepart of the present invention.

The definition of “Cop 1-related peptide or polypeptide” according tothe invention is meant to encompass other synthetic amino acidcopolymers such as the random four-amino acid copolymers described byFridkis-Hareli et al., 2002 (as candidates for treatment of multiplesclerosis), namely copolymers (14-, 35- and 50-mers) containing theamino acids phenylalanine, glutamic acid, alanine and lysine (polyFEAK), or tyrosine, phenylalanine, alanine and lysine (poly YFAK), andany other similar copolymer to be discovered that can be considered auniversal antigen similar to Cop 1.

In another embodiment, the present invention relates to the treatment ofa neurodegenerative disease or disorder selected from Huntington'sdisease, Alzheimer's disease or Parkinson's disease, which comprisesadministering to a patient in need T cells which have been activatedpreferably in the presence of Cop 1, or by a Cop 1-related peptide orpolypeptide. Such T cells are preferably autologous, most preferably ofthe CD4 and/or CD8 phenotypes, but they may also be allogeneic T cellsfrom related donors, e.g., siblings, parents, children, or HLA-matchedor partially matched, semi-allogeneic or fully allogeneic donors. Tcells for this purpose are described in U.S. Ser. No. 09/756,301 andU.S. Ser. No. 09/765,644, corresponding to WO 01/93893, each and all ofthem hereby incorporated by reference in its entirety as if fullydisclosed herein.

The dosage of Cop 1 to be administered will be determined by thephysician according to the age of the patient and stage of the diseaseand may be chosen from a range of 1-80 mg, preferably 20 mg, althoughany other suitable dosage is encompassed by the invention. The treatmentshould be preferably carried out by administration of repeated doses atsuitable time intervals, preferably every 1, 4 or 6 weeks, but any othersuitable interval between the immunizations is envisaged by theinvention according to the neurodegenerative disease to be treated, theage and condition of the patient.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The carrier(s) mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof.

For the purposes of the present invention, the composition comprisingCopolymer 1 or a Copolymer 1-related peptide or polypeptide isadministered in a regimen that confers protective autoimmunity and issometimes referred to herein as a vaccine for neuroprotectivevaccination. Such a vaccine, if desired, may contain Copolymer 1emulsified in an adjuvant suitable for human clinical use.

Thus, according to the present agent, the active agent may beadministered without any adjuvant or it may be emulsified in an adjuvantsuitable for human clinical use. The adjuvant is selected from aluminumhydroxide, aluminum hydroxide gel, and aluminum hydroxyphosphate, or anyother adjuvant that is found to be suitable for human clinical use. In apreferred embodiment, the vaccine adjuvant is amorphous aluminumhydroxyphosphate having an acidic isoelectric point and an Al:P ratio of1:1 (herein referred to as Alum-phos). It is clear that this is given byway of example only, and that the vaccine can be varied both withrespect to the constituents and relative proportions of theconstituents.

Methods of administration include, but are not limited to, parenteral,e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal(e.g., oral, intranasal, buccal, vaginal, rectal, intraocular),intrathecal, topical and intradermal routes. Administration can besystemic or local.

According to the present invention, Cop 1 or a Cop 1-related peptide orpolypeptide may be used as a sole therapy or in combination with one ormore drugs for the treatment of Alzheimer's, Huntington's or Parkinson'sdisease. When administered together with another drug or drugs suitablefor treatment of Alzheimer's, Huntington's or Parkinson's disease, theadditional drug or drugs is/are administered at the same day ofvaccination, and daily or at any other interval thereafter, according tothe manufacturer's instructions, with no association to the vaccineregimen.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Section I: Vaccination With Cop 1 for Treatment of Huntington'SDisease

Various mouse models for Huntington's disease have been establishedwhich enable the exploration of early pathological, molecular andcellular abnormalities produced by the CAG mutation. The HD R6/2transgenic mice model was selected as the in vivo test system in thepresent invention. These mice overexpress exon 1 of the humanHuntington's disease gene with an increased CAG repeat length thatencodes huntingtin (Mangiarini et al., 1996). HD R6/2 transgenic miceshow behavioral-motor deficits at as early as 5-6 weeks of age.Behavioral anomalies do not appear until 8 weeks, followed by thedevelopment of a progressive severe neurological phenotype with lowweight, clasping, tremor and convulsions, and an early death at 10-14weeks (Carter et al., 1999).

Based on the glutamate toxicity model, an optimal neuroprotective effectin mice was established by a regimen of repeated injections of 75 μg Cop1 at 4 weeks interval. The same regimen of treatment was foundbeneficial to HD R6/2 transgenic mice and reduced the rate of motorfunction deterioration, as shown by a significant preservation of therotarod performance and prolonged life span of the animals.

Materials and Methods—Section I

(i) Animals. Mice of the C57BL/6J strain, aged 8-13 weeks, were suppliedby the Animal Breeding Center of The Weizmann Institute of Science(Rehovot, Israel). Prior to their use in the experiments, the mice wereanesthetized by intraperitoneal administration of 80 mg/kg ketamine and16 mg/kg xylazine. Transgenic R6/2 mice overexpressing the human geneencoding huntingtin were obtained from the Jackson Laboratory. Allanimals were handled according to the regulations formulated by theInstitutional Animal Care and Use Committee (IACUC).

(ii) Reagents. Copolymer 1 (median MW: 7,200 dalton) was supplied byTeva Pharmaceutical Industries Ltd. (Petach Tikva, Israel).

(iii) Immunization. For immunization, Cop 1 dissolved inphosphate-buffered saline (PBS; 100 μl) was injected subcutaneously (SC)at one site in the flank of the mice. Control mice were injected withvehicle only.

(iv) Glutamate injection. The right eye of an anesthetized C57B BL/6Jmouse was punctured with a 27-gauge needle in the upper part of thesclera, and a 10-μl Hamilton syringe with a 30-gauge needle was insertedas far as the vitreal body. Mice were injected intraocularly with atotal volume of 1 μl(200 nmol) of L-glutamate dissolved in saline.

(v) Labeling of retinal ganglion cells (RGC) in mice. RGCs were labeled72 hours before the end of the experiment. Mice were anesthetized andplaced in a stereotactic device. The skull was exposed and kept dry andclean. The bregma was identified and marked. The designated point ofinjection was at a depth of 2 mm from the brain surface, 2.92 mm behindthe bregma in the anteroposterior axis and 0.5 mm lateral to themidline. A window was drilled in the scalp above the designatedcoordinates in the right and left hemispheres. The neurotracer dyeFluoroGold (5% solution in saline; Fluorochrome, Denver, CO) was thenapplied (1 μl, at a rate of 0.5 μl/min in each hemisphere) using aHamilton syringe, and the skin over the wound was sutured. Retrogradeuptake of the dye provides a marker of the living cells.

(vi) Assessment of RGC survival in mice. Mice were given a lethal doseof pentobarbitone (170 mg/kg). Their eyes were enucleated and theretinas were detached and prepared as flattened whole mounts inparaformaldehyde (4% in PBS). Labeled cells from 4-6 selected fields ofidentical size (0.5 mm²) were counted. The selected fields were locatedat approximately the same distance from the optic disk (0.3 mm) toovercome the variation in RGC density as a function of distance from theoptic disk. Fields were counted under the fluorescence microscope(magnification× 900) by observers blinded to the treatment received bythe mouse. The average number of RGCs per field in each retina wascalculated. The effectiveness of the different vaccine formulations inprotecting neurons is measured by counting the surviving RGCs.

EXAMPLE 1 Glutamate Toxicity—an In Vivo Model for Selection of Dose andRegimen of Cop 1 Vaccination

Glutamate is an amino acid normally present at low concentrations in theCNS, where it serves as the principal excitatory neurotransmitter.However, in many neurodegenerative diseases, glutamate levels rise totoxic levels, causing cell damage. This model was therefore chosen toestablish Cop 1 neuroprotective vaccination and optimize the therapeuticregimen. Glutamate toxicity is assessed by intraocular injection ofglutamate into the eyes of C57B1/6J mice and then measuring thesubsequent death of RGCs, the neurons that carry visual signals to thebrain.

1a. Cop 1 Dose Determination

To study the effect of the dose of Cop 1 vaccination onglutamate-induced RGC death, Cop 1 emulsified in complete Freund'sadjuvant (CFA; 25, 75 or 225 μg Cop 1 in total volume of 100 μl) wasinjected subcutaneously at one site in the flank of C57BL/6J mice, andseven days later glutamate (200 nmol) was injected into the vitreal bodyof the mice. After seven days, the surviving RGCs were counted. Theamount of RGCs death following glutamate toxicity without any priorimmunization was taken as 100% of protectable cells. The results,presented in FIG. 1, show that effective vaccination was obtained bytreatment with either 25 μg or 75 μg Cop 1.

Latency of neuroprotective effect was determined by vaccination with 75μg Cop 1 seven, fourteen and twenty-eight days prior to glutamateinjection. As can be seen in FIG. 2, the neuroprotective effect of asingle injection of Cop 1 is optimal at 7 days post-immunization(reduction of RGC death by ≧40%). The neuroprotective effect was reduced14 and 28 days after vaccination.

1b. Optimal Regimen of Repeated Cop 1 Injections

In an attempt to maintain the neuroprotective effect of Cop 1immunization, repeated injections of Cop 1 were evaluated. The aim wasto determine the optimal regimen of repeated Cop 1 injections that willmaximize the long term RGCs survival effect.

Cop 1 was originally developed as a therapy for multiple sclerosis (MS),an autoimmune disease characterized by unregulated T-cell activityagainst self-peptides of the CNS. Cop 1 is given to MS patients once aday at a dosage of 20 mg per patient by subcutaneous injections. Weexamined if daily injections of Cop 1 repeated for several days canmaintain the neuroprotective effect on RGCs. Mice were immunized withCop 1 daily for two or three days (Cop 1, 25 μg/mouse and 75 μg/mouse).The results, presented in FIG. 3, show that daily injections of Cop 1repeated for two days, give neuroprotection on RGCs and betterprotection is achieved with 75 μg Cop 1, while immunization during threeconsecutive days cause loss of the neuroprotective effect on RGCs.

To determine the vaccination regimen (best time interval) that producesthe optimal degree of neuroprotection, three repeated Cop 1 injectionswere administered to mice at different time intervals ranging from dailyto monthly. In one experiment, the mice received two 75 μg Cop 1injections at intervals of 1, 2, 3, 4, 6 and 8 weeks. In anotherexperiment, the mice received three repeated 75 μg

Cop 1 injections daily or at intervals of 1, 2, and 4 weeks. The resultsare shown in FIGS. 4 and 5, respectively. The neuroprotective effect ofthe treatment is represented as % of a single injection of Cop 1 (75μg/mouse) injected 7 days before glutamate toxicity was induced. Thissingle injection was determined as positive control and was performed ineach experiment. As shown in FIGS. 4 and 5, a 4-week interval betweenCop 1 injections (75 μg/mouse) had the highest neuroprotective efficacy.It is striking that daily administration of Cop 1, the regimen used astherapy for multiple sclerosis, provides poor neuroprotection.

The results using the glutamate toxicity model showed that the regimenof repeated injections of Cop 1 may lead to a sustained neuroprotectiveeffect. Based on these results, the optimal neuroprotective effect inmice was found to be repeated 75 μg injections of Cop 1 at 4 weekintervals.

EXAMPLE 2 Correlation between the Cellular Immune Response to Cop 1Vaccination and the Neuroprotective Effect

Two ex vivo markers correlate with the efficacy—the T cell stimulationindex and interferon-γ (IFN-γ). The stimulation index indicates theextent to which Cop-1-responsive T cells are present in the lymphocytepopulation. IFN-γ secretion is characteristic of T cells of the “Th1”subtype. These markers thus provide a means of profiling the cellularimmune response.

The correlation between the neuroprotective effect and the cellularimmune response to Cop 1 vaccination was thus determined by in vitroevaluation of T-cell proliferation and the level-profile of cytokinesecretion.

The effect of Cop 1 vaccination was examined by isolating spleniclymphocytes from mice immunized with different doses of Cop 1 (25, 75and 225 μg/mouse), 7, 14, 21 and 28 days after immunization, andmeasuring the proliferative response of the splenocytes to Cop 1 by[³H]thymidine incorporation, and the induction of cytokine production(IFN-γ) by ELISA assay.

Uptake of labeled thymidine by splenocytes represents proliferation ofspecific T-cells to Cop 1, following Cop 1 vaccination. The results inFIG. 6 are expressed as stimulation index (SI), where SI is the mean cpmof cells incubated in vitro with the antigen (Cop 1) divided by the meancpm of cells incubated in vitro without the antigen (Cop 1). A positiveresponse was defined as SI>2. A single injection of Cop 1 resulted inincreased SI after 7 days for the three doses. After 14 days, onlymarginal proliferation of T-cells was seen for injection of 25 and 225μg Cop 1 and less proliferation for injection of 75 μg Cop 1. The SIdecreased after 21 and 28 days, meaning that the splenocytesproliferation in response to Cop 1 had abated. These results corroboratethe glutamate toxicity results that showed that the neuroprotectiveefficacy decreases with time.

Secretion of INF-γ by stimulated splenocytes was measured by ELISA (R&DSystems). As shown in FIG. 7, the highest level of INF-γ secretion fromsplenocytes was observed 7 days after Cop 1 immunization (25 and 75μg/mouse). The levels of INF-γ declined after 14, 21 and 28 days. Theseresults are in agreement with the results obtained for neuroprotectiveefficacy and T-cell proliferation.

Neuroprotective efficacy was correlated with IFN-γ secretion, similar tothe effect shown in FIG. 1. In contrast, T-cell proliferation remainshigh under daily injections of Cop-1. This result shows that while Cop-1responsive T cells are still present, the loss of IFN-γ secretionindicates a shift in the predominant phenotype of the cells; this isaccompanied by a loss of neuroprotective efficacy. It therefore appearsthat neuroprotection is associated with IFN-γ secretion.

The above animal results are in line with the observation that dailyinjections of Copaxone® to MS patients leads to a Th2 type response(Vieira et al., 2003). Thus, the daily regimen of Cop-1 should not beexpected to confer neuroprotection and is not the regimen of choice forHuntington's disease; vaccinations spaced at wider intervals are morelikely to prove effective.

EXAMPLE 3 In Vivo Animal Test System for Huntington's Disease

The beneficial effect of Cop 1 vaccination was examined for exertion ofneuroprotective effects using the HD R6/2 transgenic mice test system.R6/2 transgenic mice over express the mutated human huntingtin gene thatincludes the insertion of multiple CAG repeats (Mangiarini et al.,1996). These mice show progressive behavioral-motor deficits starting asearly as 5-6 weeks of age, and leading to premature death at 10-13weeks. The symptoms include low body weight, clasping, tremor andconvulsions (Carter et al., 1999).

Two different doses of Cop 1 vaccination were tested, 75 μg Cop 1/mouse(n=13) and 150 μg Cop 1/mouse (n=11), that were injected when the micewere 45 days old and every 4 weeks thereafter. A third group (n=12) wasvaccinated with 75 μg Cop 1/mouse on day 60 of age and every 4 weeksthereafter. The control group (n=17) was injected with PBS starting onday 45 of age and every 4 weeks thereafter. Motor neurological functionswere evaluated using the rotarod performance test, which assesses thecapacity of the mice to stay on a rotating rod. For this test, mice wereplaced on a rod rotating at 2 rpm: the time until the mouse falls offthe rotating rod (best of three attempts, up to 180 sec for each trial),is used as the measure of animal motor-function. Each mouse was testedtwice weekly and the two scores averaged.

The results are shown in FIG. 8. Each point on the graphs represents theaverage group score for each week (SEM indicated by error bar). Thearrows on the x-axis represent the timing of Cop 1 (or PBS) injections.The results show that vaccination with Cop 1, either 75 μg/mouse or 150μg/mouse, starting on day 45 of age, produced a significant improvementin motor performance during the follow-up period of 8 to 14 weeks.However, vaccination with 75 μg Cop 1/mouse starting on day 60 of agehad no significant effect (data not shown).

Control and Cop 1 vaccinated HD R6/2 transgenic mice (150 μg/mouse) weresubjected to rotarod performance test on day 45 using four differentspeeds: 2, 5, 15 and 25 rpm. FIG. 9 shows that the improvement inrotarod performance following Cop 1 vaccination is dependent on thespeed of rotation. Significant better performance of the twelve-week oldvaccinated HD R6/2 mice compared to non-treated HD R6/2 control mice wasmost clearly apparent using 5 rpm rotarod speed.

The effect of Cop 1 vaccination on weight loss of HD R6/2 transgenicmice was tested on the three groups. Mice were weighed twice a week atthe same time during the day. No effect on body weight was observedfollowing vaccination on day 45 or day 60 using either 75 μg/mouse or150 μg/mouse Cop 1 compared to the control group.

It could also be observed that Cop 1 vaccination significantly delayedmortality and onset of disease of HD R6/2 mice. The effect of Cop 1vaccination on survival of the HD transgenic mice is shown in Table 1.Statistical comparisons of survival were made by ANOVA followed by theFisher's least significant difference test.

TABLE 1 Effect of Cop 1 vaccination on survival of HD R6/2 mice Cop 1Cop 1 Cop 1 75 μg/mouse 150 μg/mouse 75 μg/mouse Control day 45 day 45day 60 Survival 103 ± 2.5 110 ± 2.7* 101 ± 3.5 108 ± 1.6 (days) Onset of 78 ± 3.8 89 ± 4.5  91 ± 3.7*  79 ± 5.6 disease (p = 0.065)

In conclusion, the results of Examples 1 and 2 show that Cop 1vaccination attenuates neuronal cell death induced by exposure toelevated levels of the excitotoxic neurotransmitter glutamate, and thatthe neuroprotective effect is dependent upon activation andproliferation of T-cells specific to Cop 1 that secrete INF-γ (Th1). Theneuroprotective effect is short-lived, unless maintained by a boostingregime—it is build up by 7 days post immunization, and is then reduceddue to activation of regulatory cells which terminate the response. TheCop 1 dose found to be the most active in the animal models was 75 μgCop 1/mouse, that translate to a human adult dose of 20 mg on a mg/m²basis. The optimal regimen for neuroprotection in mice was monthlyinjection, both in the glutamate toxicity model and to reduce the rateof motor function deficits and to improve life expectancy in the HD R6/2transgenic mice, as shown in Example 3. Thus, for human use,neuroprotective Cop 1 vaccination should be administered in doses spacedat least one month apart, preferably 4-6 weeks apart, more preferablyevery 5 or 6 weeks.

EXAMPLE 4 Human Clinical Trials for Huntington's Disease

The primary objective of the human study is to evaluate thetolerability, safety and immunological response of the serialadministration of 20 mg or 2×20 mg dose of Cop 1 (Copaxone® or anotherCop 1 formulation) versus placebo, in patients suffering fromHuntington's disease. The secondary objective of the study is toevaluate the neurological course of patients with HD disease followingadministration of Cop 1, by measuring the following neurologicalclinical parameters: Unified Huntington's Disease Rating Scale (UHDRS)and Total Motor Scale (TMS).

Eligible patients (female and male, 18-70 years old, symptomaticpatients with clinically diagnosed HD and a confirmatory family historyof HD) will receive one administration of placebo (40 mgmannitol/injection) and three administrations of Copaxone® (20 mg/mlsubcutaneously or 2×20 mg/ml subcutaneously, 1 in each arm) at 6 weeksintervals between administrations. Blood samples for immunologicalprofile analysis will be taken at screening and prior to firstinjection. Each administration of Copolymer 1 will be followed by aseries of blood sampling to determine the immunological profile on days7, 14, 28 and just prior to next injection and termination.

UHDRS is a research tool that has been developed by the Huntington StudyGroup (HSG). The purpose of the scale is to allow the researchers tograde the symptoms of HD in a way that allows them to make accuratecomparisons between individual patients, and to better chart the courseof the disease in patients. The scale is divided into a number ofdifferent subscales, including the Total Motor Score 4 (TMS-4). In thehuman trial, a primary end-point is the change over a period of time,e.g. one-year period, in the TMS-4 subscale of the UHDRS, the standardrating scale for trials in HD. The pre-determined and end-points of thetrial (such as UHDRS scores) are compared for the patients on Copaxoneand the one may assume the possibility that the drug can be said to havehad some kind of impact on Huntington's disease.

Section II: Vaccination with Autoantigen or Cop 1 Protects Againstβ-Amyloid and Glutamate Toxicity

Neurodegenerative diseases differ in etiology but are propagatedsimilarly. In the experiments in this section, we show that neuronalloss caused by intraocular injection of aggregated β-amyloid wassignificantly greater in immunodeficient mice than in normal mice. Theneurodegeneration was attenuated or augmented by elimination oraddition, respectively, of naturally occurring CD4⁺CD25⁺ regulatory Tcells (Treg). Vaccination with retina-derived antigens or withCopolymer-1, but not with β-amyloid, reduced the ocular neuronal loss.In mouse hippocampal slices, microglia encountering activated T cellsovercame the cytotoxicity of aggregated β-amyloid. These findingssupport the concept of “protective autoimmunity”, show that a given Tcell-based vaccination is protective at a particular site irrespectiveof toxicity type, and suggest that locally activated T cells induce amicroglial phenotype that helps neurons withstand the insult Alzheimer'sand other neurodegenerative diseases might be arrested or retarded byvaccination with Cop-1 or related compounds or by treatment withcompounds that weaken Treg suppression.

In the experiments below, we showed that intraocular injection of theaged (aggregated) form of the β-amyloid peptide 1-40 (Aβ₁₋₄₀) causesloss of retinal ganglion cells (RGCs), similarly to the effect of toxicconcentrations of glutamate. We further showed that in both cases thedestructive effect could be attenuated either by elimination ofnaturally occurring CD4⁺CD25⁺ regulatory T cells (Treg) or by evoking animmune response directed against antigens derived from the tissue's ownconstitutively expressed proteins (rather than against the threateningcompound itself). The therapeutic effect could be reproduced by passivetransfer of T cells directed against the same self-antigens.

Materials and Methods—Section II

(vii) Animals. Mice were handled according to the Association forResearch in Vision and Ophthalmology (ARVO) resolution on the use ofanimals in research. Male C57BL/6J wild type, BALB/c/OLA wild type, andnude mice, all specific pathogen-free and aged between 8 and 13 weeks,were supplied by the Animal Breeding Center of The Weizmann Institute ofScience (Rehovot, Israel) under germ-free conditions. The mice werehoused in a light- and temperature-controlled room and matched for agein each experiment. Mice were anesthetized by i.p. administration ofketamine (80 mg/kg; Ketaset, Fort Dodge, Iowa) and xylazine (16 mg/kg;Vitamed, Ramat-Gan, Israel). Prior to tissue excision, the mice werekilled with a lethal dose of pentobarbitone (170 mg/kg; C.T.S., KiryatMalachi, Israel).

(viii) Antigens. Bovine interphotoreceptor retinoid-binding protein(IRBP) was purified from retinal extracts by affinity chromatography onCon A as described (Pepperberg et al., 1991). Bovine S-antigen(arrestin) was prepared from the Con A column flowthrough by the methodof Buczylko and Palczewski (Palczewski et al., 1994) as modified by Puiget al. (1995). Whole retinal homogenate (WRH) was prepared fromsyngeneic retinas homogenized in PBS. Ovalbumin (OVA), Con A, andβ-amyloid peptide 1-40 (Aβ₁₋₄₀)) were purchased from Sigma-Aldrich, St.Louis, Mo. The Aβ(₁₋₄₀) peptide was dissolved in endotoxin-free water,and β-amyloid aggregates were formed by incubation of Aβ(₁₋₄₀), asdescribed (Ishii et al., 2000). Glatiramer acetate (Copaxone®; Cop-1)was purchased from Teva Pharmaceuticals Ltd. (Petach Tikva, Israel).

(ix) Immunization. Adult mice were immunized with IRBP (50 μg),S-antigen (50 μg), Aβ₁₋₄₀ (50 μg), WRH (600 μg), or Cop-1 (75 μg), eachemulsified in an equal volume of CFA (Difco, Detroit, Mich.) containingMycobacterium tuberculosis (5 mg/ml; Difco). The emulsion (total volume0.15 ml) was injected s.c. at one site in the flank. Control mice wereinjected with PBS in CFA or with PBS only.

(x) Labeling of RGCs in mice. Labeling was carried out as described inMaterials and Methods, Section I (v).

(xi) Induction of toxicity by injection of glutamate or aggregatedAβ₁₋₄₀. The right eyes of anesthetized C57BL/6J or BALB/c/OLA mice werepunctured with a 27-gauge needle in the upper part of the sclera and aHamilton syringe with a 30-gauge needle was inserted as far as thevitreal body. Each mouse was injected with a total volume of 1 μl of PBScontaining L-glutamate (400 nmol; Sigma-Aldrich) or aggregated Aβ₁₋₄₀(50 μM; Sigma-Aldrich).

(xii) Assessment of retinal ganglion cell survival. At the end of theexperimental period the mice were given a lethal dose of pentobarbitone(170 mg/kg). Their eyes were enucleated and the retinas were detached,prepared as flattened whole mounts in 4% paraformaldehyde in PBS, andlabeled cells from four to six fields of identical size (0.076 mm²) werecounted (Schori et al., 2001b). The average number of RGCs per field wascalculated for each retina. The number of RGCs in the contralateral(uninjured) eye was also counted, and served as an internal control.

(xiii) In-situ detection of cell death by terminal deoxynucleotidyltransferase DNA (TUNEL). Mice were killed 48 h after intraocularglutamate injection and their eyes were removed and processed forcryosectioning. Frozen sections were fixed in 3.7% formalin for 10 minat room temperature and washed twice with PBS. The sections weretransferred to 100% methanol for 15 min at −20° C., washed twice for 5min in ethanol 100%, 95% and 70% successively, and then incubated for 10min with PBS. For permeabilization, proteases were digested withproteinase K for 20 min at room temperature. The open ends of the DNAfragments were labeled using an in-situ apoptosis detection kit (R&DSystems, Minneapolis, Minn.) according to the manufacturer'sinstructions. The labeled ends were detected using the fluoresceindetection kit supplied with a streptavidin-fluorescein conjugate. Thefluorescein-stained cells were visualized using a fluorescencemicroscope.

(xiv) Preparation of splenocytes depleted of CD4⁺CD25⁺ regulatory Tcells. Splenocytes prepared by a conventional procedure were incubatedwith rat anti-mouse phycoerythrin(PE)-conjugated CD25 antibody, and thiswas followed by incubation with anti-PE beads (Becton-Dickinson, BactlabDiagnostic, Haifa, Israel). After being washed, the splenocytes weresubjected to AutoMacs (Miltenyi Biotec, Bergisch Gladbach, Germany) withthe “deplete sensitive” program. Recovered populations were analyzed byFACSsort (Becton Dickinson, Franklin Lakes, N.J.) (Kipnis et al.,2002a).

(xv) Preparation of activated naïve T cells. Lymph nodes (axillary,inguinal, superficial cervical, mandibular, and mesenteric) and spleenswere harvested and mashed. T cells were purified (enriched by negativeselection) on T cell columns (R&D Systems). The enriched T cells wereincubated with anti-CD8 microbeads (Miltenyi Biotec), and negativelyselected CD4⁺ T cells were incubated with PE-conjugated anti-CD25antibodies (30 μg/10⁸ cells) in PBS/2% fetal calf serum. They were thenwashed and incubated with anti-PE microbeads (Miltenyi Biotec) andsubjected to magnetic separation with AutoMACS. The retained cells wereeluted from the column as purified CD4⁺CD25⁺ cells (Treg). The negativefraction (effector T cells, Teff), consisting of CD4⁺CD25⁻ T cells, wasfurther activated for 4 days, in medium containing 5×10⁵ cells/ml, withspleen-derived APC (irradiated with 3000 rad), and 0.5 μg/ml anti-CD3antibodies, supplemented with 100 units of mouse recombinant IL-2(mrIL-2; R&D Systems).

(xvi) Preparation of antigen-specific activated lymphocytes fromimmunized mice. Ten days after immunization, the mice were killed andtheir draining lymph nodes were excised and pressed through a fine wiremesh. The washed lymphocytes (2×10⁶ cells/ml) were activated with therelevant antigens (IRBP₁₋₂₀ or aggregated Aβ₁₋₄₀, each at 10 μg/ml) instimulation medium containing RPMI supplemented with L-glutamine (2 mM),2-mercaptoethanol (5×10⁻⁵ M), penicillin (100 IU/ml), streptomycin (100IU/ml), and autologous mouse serum 1% (vol/vol). After incubation for 48h at 37° C., 90% relative humidity, and 7% CO₂ the lymphocytes werewashed with PBS, counted, and injected intraperitoneally into autologousmice not more than 1 h after intravitreal injection of a toxic dose (50μM) of aggregated Aβ₁₋₄₀.

(xvii) Microglial cultures. Microglia were purified from the cerebralcortices of newborn (day 0) BALB/c/OLA mice, as described (Butovsky etal., 2001). IFN-γ (20 ng/ml; R&D Systems), β-amyloid (Sigma-Aldrich;aggregated Aβ₁₋₄₀25 μM), or activated T cells (1.5×10⁵ per well) wereadded to the culture medium for 12 h. After treatment, microglia werewashed three times with PBS and prepared for application on hippocampalslices.

(xviii) In-vitro model of hippocampal slices. BALB/c/OLA mice, aged 8-10days, were decapitated and their brains were rapidly removed understerile conditions and placed in ice-cold preparation medium consistingof minimum essential medium (MEM; Gibco, Carlsbad, Calif.) with 1%L-glutamine (Gibco) at pH 7.35. The frontal pole was removed and thebrains were cut into 350-μm horizontal slices on a vibratome (Pelco,Redding, Germany), beginning at the ventral surface. Slices containingthe hippocampi were cultured on Falcon cell culture inserts, pore size0.4 μm (Becton Dickinson), in 6-well plates. The cultivation mediumcontained 50% MEM, 25% Hanks balanced salt solution (Gibco), 25% normalhorse serum, 2% glutamine, 10 μg/ml insulin-transferrin-sodium selenitesupplement (Boehringer Mannheim, Mannheim, Germany), 2.64 mg/ml glucose(Braun, Melsungen, Germany), 0.1 mg/ml streptomycin, 100 U/mlpenicillin, and 0.8 μg/ml vitamin C (all from Sigma-Aldrich). Theorganotypic hippocampal slice cultures (OHSCs) were incubated at 35° C.in a humidified atmosphere with 5% CO₂ for 24 h, during which time theslices were either left untreated or treated with 4×10⁵ microglia perwell. Tissue loss was assessed by addition of propidium iodide (PI) (5μg/ml; Sigma) to the medium for 30 min at the end of the incubationperiod. Excess PI was then washed away with cultivation medium, and theslices were prepared for microscopy and visualized. To quantify neuralcell death in the OHSCs, PI intensity in each slice was assessed by useof Image-Pro software (Media Cybernetics, Carlsbad, Calif.). PI stainingintensity for a specific treatment was compared to that of the untreatedcontrol, using a two-tailed Student's t-test.

EXAMPLE 5 Retinal Proteins can Evoke a Protective T Cell-Based Responseto Glutamate Intoxication

We have shown previously that mice of different genetic backgroundsdiffer in their ability to resist injurious conditions (Schori et al.,2001b; Kipnis et al., 2001; Schori et al., 2002). The differences wereattributed, at least in part, to strain-related variations in theability to manifest a T cell-dependent protective response (Kipnis etal., 2001). In view of the observed failure of myelin proteins toprotect mice against glutamate toxicity in the eye and the successfulprotection against glutamate toxicity in rats by retinal proteins(Schori et al., 2001b; Mizrahi et al., 2002) we were interested inexamining whether immunization of mice with retinal proteins wouldimprove their neuronal survival after exposure to glutamate toxicity,and if so, whether the same vaccination would be effective against otherthreatening compounds (such as aggregated β-amyloid) injected into thesame site. Glutamate (400 nmol) was injected into the right eyes ofC57BL/6J mice, and 48 h later we examined retinal cryosections subjectedto terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL). Apoptotic cell death was observed in the RGC layer (FIG. 10A).When mice of this strain were vaccinated with a homogenate of wholeretinal proteins (WRH) in CFA 6 days before being injected with a toxicdose of intraocular glutamate (400 nmol), examination 1 week laterdisclosed survival of significantly more RGCs than that seen in age- andstrain-matched control mice injected with PBS emulsified in CFA(2239.7±153.3 and 1480.6±176.2, respectively, P<0.0001; two-tailedStudent's t-test). This finding indicated that vaccination with theretinal components had increased the ability of the immunized mice towithstand glutamate toxicity. FIG. 10B shows that the vaccinationsignificantly reduced RGC loss, expressed as a percentage of the numbersin normal controls (mean±SEM; FIG. 10B).

In light of the above results, and to further test our hypothesis thatthe evoked protection against glutamate toxicity is an outcome of a Tcell-mediated response to the retinal self-antigens, we examined whetherimmunization with the specific eye-resident antigens interphotoreceptorretinoid-binding protein (IRBP) or S-antigen (retinal arsenin), ratherthan with the retinal homogenate, can protect RGCs against glutamatetoxicity Immunization of C57BL/6J mice, 10 days before glutamateinjection, with IRBP emulsified in CFA, resulted in survival ofsignificantly more RGCs than in glutamate-injected C57BL/6J miceimmunized with PBS/CFA (1996±49.53 and 1649±43, respectively, P<0.0008;two-tailed Student's t-test; FIG. 10C) Immunization with the retinalself-antigen S-antigen in CFA resulted in a similar increase in neuronalsurvival relative to immunization with PBS/CFA (2160±38 and 1648±37,respectively, P<0.0001; two-tailed Student's t-test; FIG. 10D). For easeof comparison, the results in the figure are presented as the loss ofneurons expressed as a percentage of the number of RGCs instrain-matched normal retinas.

It should be emphasized that the retinal self-proteins IRBP andS-antigen, both of which are capable of causing uveitis in susceptiblemice (Caspi et al., 1990a; Caspi et al., 1990b) but were used here forpurposes of protection, are not intended for development as atherapeutic vaccination; this is purely an experimental paradigm, usedhere for proof of concept, that supports our previous contention thatthe same T cells can be both protective and destructive, and that theiractual effect is a reflection of the tissue context, the quantity of Tcells, and the timing of their activities in the tissue (Mizrahi et al.,2002; Hauben et al., 2001; Fisher et al., 2001).

EXAMPLE 6 Ability to Withstand the Toxicity of β-Amyloid is TCell-Dependent

Having shown that the physiologically relevant antigen for protectionagainst neurotoxicity is not the toxic compound itself (glutamate) but aself-antigen that resides in the site of damage, we then examinedwhether the same vaccination might be beneficial against different toxicself-compounds provided that the toxicity is restricted to the samesite. We tested this hypothesis by examining the effect of thevaccination on the toxicity of aggregated β-amyloid. Aggregatedβ-amyloid (Aβ₁₋₄₀ (5 or 50 μM) was injected into the right eyes ofC57BL/6J mice. This model (intraocular injection of β-amyloid) waschosen not because of the supposed association of this compound with theoptic neuropathy in Alzheimer's disease (Bakalash et al., 2002;Schwartz, 2004), but because β-amyloid is capable of causing RGC death(Jen et al., 1998), and therefore its use allows us to further explorethe concept of antigenic specificity. Surviving RGCs were counted 1 or 2weeks after ocular injection of aggregated β-amyloid. After 1 week, thenumbers of viable RGCs were 2257±77 (5 μM injection) and 2071±30 (50 μMinjection), and after 2 weeks they were 2062±41 (5 μM) and 1952±21 (50μM). A total of 3445±57 neurons were counted in naïve mice. Under thesame experimental conditions, toxicity caused by injection of thevehicle alone did not affect more than 5% of RGCs in the normal retina.FIG. 11A shows the β-amyloid-induced neuronal loss expressed as apercentage of the average number of RGCs in normal wild-type retina.FIGS. 11B and 11C show representative photomicrographs of whole-mountedretinas excised from mice after intraocular injection of PBS andaggregated Aβ₁₋₄₀, respectively.

To determine whether the ability of naïve mice to withstand the toxicityof aggregated Aβ₁₋₄₀ is T cell-dependent, we compared RGC survival inwild type and nude (nu/nu) BALB/c/OLA mice 2 weeks after intraocularinjection of aggregated Aβ₁₋₄₀ (50 μM). Significantly more neuronssurvived in the injected wild-type mice (2316±53) than in their Tcell-deficient counterparts (1779±147; P<0.01). The choice of BALB/c/OLAmice for this experiment was based on a previous observation that the Tcell-dependent ability of this strain to withstand the consequences ofCNS injury is significantly better than that of C57BL/6J mice (Schori etal., 2001b; Kipnis et al., 2001; Kipnis et al., 2004), and thus anydifferences resulting from the absence of T cells would be more easilydetectable in the BALB/c/OLA mice. FIG. 11D shows neuronal loss as apercentage of the number of neurons in normal retinas. FIGS. 11E and 11Fshow representative micrographs of retinas from wild-type and nude mice,respectively, after intraocular injection of aggregated Aβ₁₋₄₀. Theseresults support the contention that the strain is a factor in theability of neural tissue to withstand ocular toxicity, and show thatstrain-related differences in that respect are related not to the typeof insult but to the ability to harness a well-controlled Tcell-dependent immune response.

To further test our working hypothesis that the T cell specificityneeded for neuroprotection is directed not against the threateningcompound but against self-antigens that reside in the site of thelesion, we subjected C57BL/6J mice to intraocular toxicity of aggregatedAβ₁₋₄₀ and then immunized them with the IRBP-derived peptide (which, asin the case of glutamate toxicity (FIG. 10C), is protective against theintraocular toxicity of aggregated Aβ₁₋₄₀). After vaccination, the lossof RGCs induced by aggregated Aβ₁₋₄₀ was significantly smaller than thatobserved in mice immunized with PBS/CFA (RGC survival was 2307±62 forIRBP/CFA and 1840±56 for PBS/CFA; FIG. 12A). We also immunized C57BL/6Jmice with the non-aggregated (non-toxic) form of the β-amyloid peptidebefore injecting them intraocularly with Aβ₁₋₄₀. After vaccination withβ-amyloid/CFA, the loss of RGCs induced by aggregated Aj3₁₋₄₀ did notdiffer significantly from that observed in mice immunized with PBS/CFA(RGC survival was 1743±55 and 1831±45, respectively; FIG. 12B).

To verify that the observed vaccination-induced protection is Tcell-dependent, we prepared primary T cells directed against IRBP andS-antigen or against IRBP only. After their activation ex vivo, thelymphocytes (1.2×10⁷ cells) were transferred into naïve C57BL/6J micefreshly exposed to toxicity of glutamate or aggregated Aβ₁₋₄₀.Significantly more RGCs survived in mice that received lymphocytesactivated with IRBP+S-antigen than in mice immunized with lymphocytesactivated by the non-CNS antigen OVA (2220±38 compared to 1652±56,P<0.001; two-tailed Student's t-test; FIG. 13A). RGC survival in micethat received OVA-activated T cells did not differ significantly fromthat in naïve mice injected with glutamate (1652.6±56 and 1535.6±74,respectively; not shown). Results are expressed as the percentageincrease in neuronal survival relative to survival in control mice (FIG.13A).

Similarly, T cells specific to IRBP or to non-aggregated Aβ₁₋₄₀ obtainedfrom immunized mice were prepared and activated ex vivo, and thenpassively transferred into C57BL/6J mice exposed to toxicity ofaggregated Aβ₁₋₄₀. Passive transfer of the T cells activated withIRBP-derived peptide was beneficial in mice that were injectedintraocularly with aggregated Aβ₁₋₄₀, as shown by the significantlysmaller loss of RGCs in these mice than in normal control mice (FIG.13B). In contrast, in mice that received a passive transfer of T cellsspecific to non-aggregated Aβ₁₋₄₀, the loss of RGCs after intraocularinjection with a toxic dose of aggregated Aβ₁₋₄₀ differed only slightlyfrom that in mice treated with PBS.

These findings confirmed that the protection achieved by activevaccination with IRBP (FIG. 12A) against the toxicity of aggregatedAβ₁₋₄₀ was T cell-mediated. The failure of T cells directed to theaggregated Aβ₁₋₄₀ itself to confer protection is in line withobservations in our laboratory that microglia, upon encounteringaggregated Aβ₁₋₄₀, fail to express MHC class II (MHC-II). Consequently,such microglia fail to present β-amyloid to the T cells, with the resultthat even if β-amyloid-specific T cells home to the CNS they will not belocally activated (Butovsky et al., unpublished observations). To fightβ-amyloid toxicity, a more appropriate choice would therefore beantigens that reside in the site and can be presented to homing T cells.

The results summarized above suggest that an appropriate choice forvaccination in order to fight β-amyloid toxicity would be antigens thatreside in the site of degeneration and that can be presented to thehoming T cells. Because of the diversity of the human histocompatibilitycomplex, vaccination with self-antigens cannot be assumed to be safe fortherapeutic purposes. In searching for a safe vaccine we examined theefficacy of vaccination with the synthetic antigen glatiramer acetate(Cop-1) (Schori et al., 2001b; Kipnis et al., 2000) which was previouslyshown to act as a partial agonist or an altered peptide ligand inmimicking the effect of a wide-range of self-reactive T cells withoutcausing an autoimmune disease (Schori et al., 2001b). Significantprotection against toxicity of aggregated β-amyloid was obtained byvaccinating C57BL/6J mice with Copolymer 1 seven days before they wereinjected intraocularly with a toxic dose of aggregated Aβ₁₋₄₀ (FIG. 14).RGC survival in Cop-1-treated and PBS-treated mice was 1939±80 and1617±43, respectively P<0.01; two-tailed Student's t-test.

EXAMPLE 7 Naturally Occurring Regulatory CD4⁺CD25⁺ T Cells Restrict theBody's Ability to Withstand β-Amyloid Toxicity in the Retina

The above observation that in the absence of intervention the ability ofmice to withstand the toxicity of aggregated Aβ₁₋₄₀ is T-cell dependent(FIG. 11) prompted us to investigate whether the ability of the neuraltissue to spontaneously withstand the toxicity of aggregated β-amyloidis suppressed by the naturally occurring regulatory CD4⁺CD25⁺ T cells(Treg), as described in the case of other CNS insults (Kipnis et al.,2002a; Schwartz and Kipnis, 2002a). If so, elimination or weakening ofsuch control might serve as an additional way to harness the autoimmuneT cells needed for protection against β-amyloid-associatedneurodegenerative conditions.

We therefore examined whether the ability of the murine neural tissue towithstand the toxicity of aggregated Aβ₁₋₄₀ could be boosted by removalof Treg. In adult C57BL/6J mice that had undergone thymectomy 3 daysafter birth (a procedure that results in Treg depletion [Sakaguchi etal., 2001; Seddon, 2000]), significantly more RGCs survived exposure toaggregated Aβ₁₋₄₀ than in matched non-thymectomized controls (2251±53and 1918±94, respectively; P<0.01; two-tailed Student's t-test; FIG.15A). In a complementary experiment, nude mice of the BALB/c/OLA strainwere replenished with 4.5×10⁷ wild-type splenocytes from which the Tregpopulation had been removed ex vivo. As controls, we used nude micereplenished with the same number of splenocytes, which were obtainedfrom whole spleens of wild-type mice and therefore contained both Tregand effector T cells (Teff). Three days after replenishment, therecipient mice were injected with a toxic dose of aggregated Aβ₁₋₄₀, andsurviving RGCs were counted 2 weeks later. Significantly fewer RGCs diedin the mice replenished with splenocytes depleted of Treg than in micereplenished with a normal splenocyte population; in both groups,however, fewer RGCs died than in the group of untreated nu/nu miceinjected with aggregated Aβ₁₋₄₀ (RGC survival was 2412±61, 2246±101, and2080±56, respectively; FIG. 15B). These findings suggest that Tregnormally down-regulate the ability of the neural tissue to spontaneouslywithstand aggregated Aβ₁₋₄₀ toxicity. PCR testing for Foxp3 expression,found to be associated with Treg (Khattri et al., 2003), confirmed thatthe Treg were Foxp3-positive whereas CD4⁺CD25⁻ T cells wereFoxp3-negative (FIG. 15C).

EXAMPLE 8 T Cells Prevent Microglia from Developing an InflammatoryCytotoxic Phenotype

We have previously proposed that one way in which the autoimmune T cellshelp to fight off destructive self-compounds is by controlling theactivity of microglia (Schwartz et al., 2003). Using organotypichippocampal slice cultures (OHSCs), our group showed that after ratmicroglia are pretreated with aggregated Aβ₁₋₄₀ they become cytotoxic toneural tissue and their ability to express MHC-II is suppressed(Butovsky et al., unpublished observations). We therefore carried out anin-vitro experiment to determine whether murine microglia exposed toaggregated Aβ₁₋₄₀ also become cytotoxic, and if so, whether activated Tcells can overcome the toxicity. After exposure of mouse microglia toaggregated Aβ₁₋₄₀, their addition to mouse OHSCs resulted insignificantly more neuronal death than that seen in OHSCs that wereuntreated or were treated with naïve microglia (FIG. 16). The loss wassignificantly reduced, however, if the added microglia, at the time oftheir exposure to aggregated Aβ₁₋₄₀, had also been exposed to activatedeffector (CD4⁺CD25⁻) T cells (FIG. 16A). Representative micrographs ofvariously treated OHSCs and untreated controls are shown in FIG. 16B(1-4). These findings support the contention that exposure of microgliato activated T cells in suitably controlled amounts not only preventsthe microglia from becoming cytotoxic, but also enables them to becomeneuroprotective. It should be noted that the microglial toxicity assayedin vitro does not reflect the lack of MHC-II expression, as thisbioassay does not require antigen presentation.

EXAMPLE 9 In Vivo Animal Test System for Alzheimer's Disease

The beneficial effect of Cop 1 vaccination can be examined for exertionof neuroprotective effects using the transgenic mice test system.

There are no spontaneous animal mutations with sufficient similaritiesto AD to be useful as experimental models. Various transgenic animalmodels for testing potential treatments for Alzheimer's disease areknown. Models are known that are based on the ability to controlexpression of one or more of the three major forms of the humanβ-amyloid precursor protein (APP), APP695, APP751, and APP770, orsubfragments thereof, as well as various point mutations based onnaturally occurring mutations, such as the familial Alzheimer's disease(FAD) mutations at amino acid 717, and predicted mutations in the APPgene, as described in U.S. Pat. No. 6,717,031 and Johnson-Wood et al.(1997). A suitable model is, for example, the transgenic hAPP770/FAD717mouse model.

To test the effect of Cop 1, a suitable formulation comprising Cop 1 isadministered to the offspring of the transgenic mice or cells derivedtherefrom, and detecting or measuring an Alzheimer's disease marker inthe transgenic mouse, or in cells derived from the transgenic mouse. Inone preferred embodiment, the Alzheimer's disease marker is a behaviorand the observed difference is a change in the behavior observed in thetransgenic mouse to which the compound has been administered. Thisbehavior may be behavior using working memory, behavior using referencememory, locomotor activity, emotional reactivity to a novel environmentor to novel objects, and object recognition. For example, the behaviorof the Cop 1-treated Alzheimer transgenic mouse model can be testedusing the Morris water maze, as described (Postina et al., 2004). It isexpected that the treated animals will exhibit an improvement in theirbehavior.

Discussion

The results of the present invention as described in Section II abovesuggest that in developing a therapeutic vaccination to counteract thetoxicity caused by accumulation of aggregated Aβ₁₋₄₀ and other toxicagents such as glutamate, the same vaccine can be used provided that thetoxic agents are all located, as is often the case, in the same site. Inthe mouse model used in Section II above, two neurotoxic self-compoundswere injected into the eye, and protection against both of them wasachieved by vaccination with the same antigens, namely peptides derivedfrom proteins that reside in the eye. We interpret this finding as proofof principle that dominant self-antigens constitutively residing in asite of damage are the self-protective antigens against threateningconditions at this site. We further show that depletion of the naturallyoccurring CD4⁺CD25⁺ regulatory T cells (Treg) can increase thespontaneous response to such antigens and thus the ability to withstandthe toxic effect of aggregated β-amyloid. As a therapeutic strategy,however, we propose vaccinating with Copolymer 1, a synthetic weakagonist of self-antigens (Schori et al., 2001b; Kipnis et al., 2000;Angelov et al., 2003; Ziemssen et al., 2002), rather than with thesite-specific self-proteins themselves, because the former can be usedas a protective vaccine without risk of inducing an autoimmune disease,a potential hazard associated with inherently inadequate control ofautoimmunity (Schwartz, and Kipnis, 2002). As an alternative strategy,we propose the use of any manipulation that will weaken the activity ofTreg (Kipnis et al., 2003).

Neurodegenerative disorders such as Parkinson's, Alzheimer's,Huntington's, prion, motor neuron diseases, and other devastatingchronic neurodegenerative syndromes have several features in common,including the accumulation of self-proteins that have either becomeaggregated or undergone conformational changes (Perlmutter, 2002). Inthe case of Alzheimer's disease, accumulation of aggregated Aβ₁₋₄₀ ispotentially a major cause of neuronal toxicity (Hardy and Selkoe, 2002).The present results support the contention that the β-amyloid peptide inits aggregated form (found in senile plaques) has a toxic effect in theCNS, not only because it is directly toxic to neurons (Jen et al., 1998;Carter and Lippa, 2001) but also because it apparently induces microgliato adopt a cytotoxic phenotype. In addition, the failure of β-amyloidvaccination to protect against β-amyloid-induced stress in the eye is inline with observations from our laboratory that cell-surface MHC-IIexpression is impaired in microglia encountering aggregated β-amyloid(Butovsky et al., unpublished observations).

In the past, it was generally assumed that because activated microgliaare seen in the context of neurodegenerative diseases, these cellscontribute to the ongoing degeneration (Qin et al., 2002). Accordingly,a substantial research effort was devoted to achieving theirsuppression.

The results in Section II above indicate that an alternative approach tothe problem necessitates modulation of the microglial phenotype, therebynot only minimizing the risk carried by malfunctioning microglia butalso exploiting microglial assistance in withstanding the destructiveeffects of aggregated Aβ₁₋₄₀ and other toxic agents associated withongoing degeneration such as glutamate and oxidative stress. Thephenotype acquired by microglia exposed to activated T cells is notdestructive insofar as it does not produce inflammation-associatedenzymes or promote redox imbalance (Schwartz et al., 2003). Thus, Tcells that can be locally activated, irrespective of the identity of theantigen(s) residing in the damaged site, can trasform the adjacentmicroglial population from an enemy into a friend.

In the experiments described in Section II above, we observedstrain-related differences in the ability of mice to withstand thetoxicity of aggregated Aβ₁₋₄₀. The present results are also in line withour contention that naturally occurring CD4⁺CD25⁺ regulatory T cellsconstitutively control the ability to withstand neurodegenerativeconditions. Although these cells are key participants in protectionagainst autoimmune disease (Kohm et al., 2002), they limit the abilityto fight degeneration in the CNS (Kipnis et al., 2002a). We havepreviously postulated that the presence of these cells reflects anevolutionary compromise between the need for autoimmune protection andthe risk of developing an autoimmune disease because of inadequatecontrol of the immune response (Kipnis et al., 2002a; Schwartz andCohen, 2000), the latter being an outcome of the failure of Treg todisplay optimal suppressive activity (Kohm et al., 2002). In rats ormice devoid of Treg, the susceptibility to autoimmune diseasedevelopment is increased, despite the benefit in terms of protectionagainst injurious conditions Therefore, one of the aims ofneuroprotective therapy is to weaken Treg. Thus, pharmacologicalintervention with a compound that mimics the physiological weakening(but not blocking) of Treg might provide a way to boost the T cell-basedself-defense.

It was shown by our group that the same autoimmune T cells can be bothsupportive and destructive (Kipnis et al., 2002b). Accordingly, inanimals that are inherently susceptible to autoimmune disease theprotocol used for eliciting the T cell response critically affects theoutcome. Thus, a strong adjuvant might lead to an autoimmune responsewhose benefit is offset by its persistence or intensity (Hauben et al.,2001). In such susceptible strains, however, autoimmune response to CNSmight not be expressed early enough to be accommodated within thetherapeutic window, or it might fail to meet other requirements, such astimely shut-off (Shaked et al., 2004a). Moreover, in susceptible strainsdevoid of immune cells (SCID) and thus lacking a T cell-based regulatorymechanism, passive transfer of encephalitogenic T cells causes EAE, butis not sufficient for conferring any neuroprotection (Kipnis et al.,2002b). In contrast, when CD4⁺CD25⁺ regulatory T cells are passivelytransferred into SCID mice (Kipnis et al., 2004b), they can have aprotective effect similar to that of encephalitogenic T cells passivelytransferred into the wild type (Hauben et al., 2000a, 2000b; Moalem etal., 1999a). In animals that are inherently resistant to autoimmunediseases the likelihood that the spontaneously evoked response to a CNSinjury will be destructive is very low; on the other hand, it might betoo weak to be beneficial and need boosting. Thus, whether or notautoimmunity will be beneficial under severe conditions in susceptiblestrain is determined by both regulation and context.

For therapies capable of meeting the criteria of both resistant andsusceptible strains without running the risk of negative side effects,the use of weak synthetic antigens such as Cop-1 or other relatedcompounds deserves consideration. Such a strategy, unlike vaccinationwith a peptide derived from a toxic antigen such as β-amyloid, canpotentially provide risk-free benefit. Moreover, the same safe antigencan be used for protection at different sites of degeneration, asituation that is often required in patients.

The results herein further support the contention that the way in whichthe body harnesses the immune system for protection againstneurodegenerative conditions is via a T cell-dependent pathway. Inaddition, they strengthen the notion that in adopting a therapeuticapproach to neurodegenerative diseases characterized by proteindeposition, the antigen selected for vaccination should not be thedisease-specific protein such as the aggregated Aβ₁₋₄₀ in Alzheimer'sdisease, Lewy bodies in Parkinson's disease, or prion protein (PrP) inprion disease (Dodart et al., 2003; White et al., 2003), but a peptidederived from an immunodominant self-protein that resides at the site ofCNS damage, a cryptic self-peptide, or an altered self-peptide, butpreferably a non-self peptide that cross-reacts weakly with self such asCopolymer 1 and Copolymer 1-related peptides and polypeptides.

The T cell-based vaccination described in Section II above protectedmice from the neurodegenerative effects of existing aggregated Aβ₁₋₄₀.The proposed strategy does not argue against the possible benefit ofantibodies specific to Aβ-amyloid (Dodart et al., 2003; Furlan et al.,2003; Mohajeri, et al., 2002) as long as the peptide used forvaccination is not encephalitogenic. The two approaches, rather thanbeing mutually antagonistic, might complement one another.

Section III: Vaccination with Cop 1 for Treatment of Parkinson'S Disease

Parkinson's disease (PD) is a neurodegenerative movement disordercharacterized by a progressive loss of dopaminergic neurons in thesubstantia nigra and depletion of the neurotransmitter dopamine in thestriatum. The best model of PD to date is the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model. MPTPhas been shown to induce parkinsonism both in man and non-humanprimates. The neurotoxicity produced by 1-methyl-4-phenylpyridinium ion(MPP⁺), a metabolite of MPTP, which is intracellularly transported intodopaminergic neurons, is thought to mimic human PD and provides a goodmodel for studying neuroprotection in PD. The beneficial effect of Cop 1immunization is examined for exertion of neuroprotective effects usingthe MPTP mice test system or another suitable model for Parkinson'sdisease. Neuroprotective therapy for PD with Cop 1 can attenuate theneurodegenerative effects and the rate of disease progression.

Materials and Methods—Section III

(xix) Animals. Male mice of the C57BL/6J strain, aged 8-13 weeks,supplied by the Animal Breeding Center of The Weizmann Institute ofScience (Rehovot,

Israel), are handled according to the regulations formulated by theInstitutional Animal Care and Use Committee (IACUC).

(xx) Reagents. Cop 1 (median MW: 7,200 dalton) is from TevaPharmaceutical Industries Ltd. (Petach Tikva, Israel). MPTP.HC1 (Sigma)is dissolved in 0.9% NaCl.

(xxi) Immunization. For immunization, Cop 1 dissolved in PBS (100 μl) isinjected SC at one site in the flank of the mice. Control mice areinjected with vehicle only.

(xxii) MPTP Treatment. Male SJL mice in different groups are treatedwith different schedules of MPTP to induce degenerative processes thatvary in intensity and time-course, for example, 18-20 mg/kg MPTP.HCl(Sigma) in PBS injected intraperitoneally (i.p.) four times, at 2 hintervals, over 1 day, or 20 mg/kg MPTP injected s.c twice per day, over2 days. Control mice are administered a corresponding volume of vehiclealone.

EXAMPLE 10 Effect of Cop 1 in the Parkinson MPTP Rodent Model

Male C57BL/6J mice are injected i.p. with 20 mg/kg MPTP.HCl in PBS, fourtimes, at 2-h intervals. Cop 1 (75 μg or 150 μg Cop 1/mouse in PBS) orPBS (control group) is administered to the MPTP-treated animals 12 hafter the last MPTP administration.

The motor dysfunction in PD is due to a profound reduction in striataldopamine content caused by the loss of dopaminergic nerve fibers in thestriatum. Motor performance of MPTP-treated mice immunized with Cop 1 orinjected with PBS is measured on a Rotarod, as previously described(Hunot et al., 2004). The rotarod performance test assesses the capacityof the mice to stay on a rotating rod. It can be expected thatimmunization with Cop 1 will display improvement of motor functions onthe Rotarod (increased Rotarod time) compared to control mice.

Other PD parameters related to neuroprotection can be carried out oneweek or more after immunization such as stereological quantification ofdopamine neuron number and optical density measurement of dopamine fiberloss using immunostaining for dopamine transporter (DAT) and tyrosinehydroxylase (TH).

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1. A method for treating a neurodegenerative disorder or disease inwhich there is accumulation of misfolded and/or aggregated proteins,selected from the group consisting of Huntington's disease andAlzheimer's disease, said method comprising administering to anindividual in need thereof an active agent selected from the groupconsisting of (i) Copolymer 1, (ii) a Copolymer 1-related peptide, (iii)a Copolymer 1-related polypeptide, and (iv) T cells activated with (i),(ii) or (iii).
 2. The method in accordance with claim 1, wherein saidactive agent is Copolymer
 1. 3. The method in accordance with claim 1,wherein said active agent is T cells which have been activated byCopolymer
 1. 4. A method for reducing disease progression, and/or forprotection from neurodegeneration and/or protection from glutamatetoxicity in a patient suffering from Alzheimer's disease, whichcomprises administering to an individual in need thereof an effectiveamount of an active agent selected from the group consisting of (i)Copolymer 1, (ii) a Copolymer 1-related peptide, (iii) a Copolymer1-related polypeptide, and (iv) T cells activated with (i), (ii) or(iii).
 5. A method for reducing disease progression, and/or forprotection from neurodegeneration and/or protection from glutamatetoxicity in a patient suffering from Huntington's disease, whichcomprises administering to said patient in need thereof an effectiveamount of an active agent selected from the group consisting of (i)Copolymer 1, (ii) a Copolymer 1-related peptide, (iii) a Copolymer1-related polypeptide, and (iv) T cells activated with (i), (ii) or(iii).
 6. A method for treatment of a patient suffering from aneurodegenerative disease or disorder selected from the group consistingof Huntington's disease and Alzheimer's disease, which comprisesimmunizing said patient with a vaccine comprising an amount of Copolymer1 effective for reducing disease progression in said patient.
 7. Amethod for treatment of a patient suffering from neurodegenerativedisease or disorder selected from the group consisting of Huntington'sdisease and Alzheimer's disease, which comprises immunizing said patientwith a vaccine comprising an amount of Copolymer 1 effective forprotection from neurodegeneration in said patient.
 8. A method fortreating or preventing neurodegeneration and cognitive decline anddysfunction associated with Huntington's disease or Alzheimer's disease,said method comprising administering to an individual in need an activeagent selected from the group consisting of (i) Copolymer 1, (ii) aCopolymer 1-related peptide, (iii) a Copolymer 1-related polypeptide,and (iv) T cells activated with (i), (ii) or (iii).
 9. The methodaccording to claim 8, wherein said active agent is Copolymer 1.